This application addresses the broad Challenge Area (06) Enabling Technologies and the specific Challenge Topic, 06-GM-105: Small RNAs. Nearly 60% of the US adult population is either obese or overweight. Clearly understanding the fundamental mechanisms of gene regulation in response to changes in energy availability is of clinical, pharmaceutical, and societal importance. The long-term objective of this research is to test the hypothesis that regulation of hypothalamic miRNA levels occurs with changes in energy availability targeting the expression of specific genes that control body weight. This multiple PI proposal is strengthened by the combined expertise of each individual laboratory. Dr. Good's lab has more than 10 years experience in studying the transcriptional control of hypothalamic genes in response to changes in energy availability. Dr. Jensen is world-renowned for his work on microarray quality and assessment of gene expression analyses. Dr. Helm's lab uses proteomic and metabolomic approaches to study cellular processes such as quiescence. Together, the three PIs will use miRNA and mRNA microarrays, proteomics and bioinformatics to ask specific and fundamental questions about the role of miRNA in body weight regulation including: (i) whether miRNA species are differentially-regulated in response to changes in energy availability (ii) if these miRNA species target specific hypothalamic genes that regulate body weight control. The co-regulated miRNA, mRNAs and proteins will be identified through a three-pronged bioinformatics approach to examine the expression levels of the putative targets of the identified miRNA species and compare these with possible discordance between the protein and mRNA levels for the target genes. This is an innovative method that has not yet been done for whole animal tissues. These studies specifically address the intent of the NIH Challenge Grants by (1) proposing to study the scientific challenge of the role of miRNA in obesity-a gap in our knowledge, especially as it relates to hypothalamic gene regulation;(2) proposing a study that is designed to benefit the scientific community through these 2-year stimulus funds, by generating at least 3 new datasets for us and others in the scientific community to use in future studies;and (3) proposing a study design with research methods that will benefit from an influx of funds to quickly advance the area of miRNA research in significant ways. With obesity at record proportions, the discovery of new mechanisms of gene regulation will lead to important new clinical, pharmaceutical and basic research directions and applications. PUBLIC HEALTH RELEVANCE: The proposed experiments will characterize a potentially new method for hypothalamic gene regulation during food intake, food deprivation or in obese individuals with high levels of circulating leptin. An understanding of these molecular pathways in the brain, and identification of the genes and proteins that are differentially regulated during changes in food availability should identify new therapeutic options for obese and overweight individuals, as well as give new information about the basic mechanisms that our brains use to control body weight.